Injectable implant of insoluble globin

ABSTRACT

The invention relates to a preparation that can be injected or implanted into the human or animal body, which comprises, as main component, globin that is insoluble at physiological pH, biocompatible and sterile.

[0001] The aim of the present invention is to provide globinpreparations that are useful for administration to humans. Thesepreparations may in particular be in the form of injectable pastes or ofimplantable solid materials, or of implants.

[0002] Many medical applications of collagen have already beendescribed, whether in the form of pastes, for example for filling, offluid or solid formulations, such as films or compresses, or in the formof diverse implants. In fact, only animal collagen is generally used.

[0003] The preparation of human collagen, which would be preferable toanimal collagen, can be envisioned from human cutaneous tissues.However, it is made very difficult since the taking of human tissuesamples from cadavers poses considerable ethical problems and requiresexpensive tests in order to eliminate the risks of transmission ofinfectious diseases, viral diseases or the like. The preparation ofhuman collagen from placentas is expensive, complex and difficult toorganize. The preparation of human collagen by the modern methods ofgenetic recombination or of cell culture is also very expensive, whichwould certainly impair the commercial development of this product.

[0004] Globin is the protein constituting hemoglobin, which, itself,contains 4 peptide chains (2 α-chains and 2 β-chains), each associatedwith a heme. The heme consists of a tetrapyrole structure containing onepositively charged iron atom. There are 4 hemes per molecule,responsible for the red coloration of hemoglobin.

[0005] The processes for preparing globin have been known for a verylong time and have been developed with dietary application being theaim, or for preparing injectable pharmaceutical solutions.

[0006] Unlike hemoglobin, which is completely soluble at physiologicalpH, globin is notably insoluble under the same conditions. The insolublenature of globin under physiological conditions has, to date, impairedthe development of its pharmaceutical applications. For this reason,most experiments have sought to prepare globin derivatives which aresoluble at physiological pH, in particular by succinylation usingsuccinic anhydride or by acetylation using acetic anhydride.

[0007] An injectable product combining a preparation of soluble globinwith insulin has been developed and marketed. It allows, afterinjection, gradual delivery of the insulin from this complex. Globin isnot the active element or the main element of this product.

[0008] The present invention proposes to provide novel materials andinjectable preparations or preparations which are implantable in theorganism, in which the globin is the main active element, and which donot have the drawbacks of the known materials and formulations, forexample of collagen or the like.

[0009] A subject of the invention is a pasty or solid preparation ofglobin that is insoluble at physiological pH, biocompatible, sterileand, preferably, biodegradable, in particular in the form of aninjectable paste, of solid materials, for example of films, or of solidimplants.

[0010] The present invention proposes to conserve the natural insolublenature of globin at neutral pH, for example by harvesting, bycentrifugation, a protein precipitate of globin formed by suspension ofthis precipitate in a pharmaceutically acceptable vehicle, for examplean aqueous physiological solution of PBS type, containing 9 g/l NaCl andbuffered at neutral pH between 6.8 and 7.5. The paste thus formed isinjectable using a fine needle (diameter 24 to 30 g). This paste can beprepared in the presence of lubricants such as polyethylene glycol,sodium hyaluronate, hyaluronic acid or other polysaccharides ormucopolysaccharides. Such an additive facilitates passage of the pastyprecipitate through a fine needle and injection thereof as implant.

[0011] The originality and the advantage of this product lie in the factthat it is a protein paste that is completely biocompatible with thesurrounding tissues into which it is injected. This protein hasundergone no alteration or chemical modification, it is naturallyinsoluble from the moment it is in a physiological environment. Aprotein paste of human globin can be used in humans for filling skincavities, wrinkles or scars, or augmenting the volume of certain tissues(urinary or digestive sphincters, vocal chords, etc.) The fillingapplications permitted by this globin paste are therefore numerous andunexpected for this protein.

[0012] The homologous human globin is preferable and makes it possibleto avoid any immunological reaction by the patient to be treated, duringor after injection. This product therefore represents a considerableadvantage with respect to the collagen which to date is prepared fromanimal skin (calf, pig, etc.) and which requires a certain number ofprecautions and conditions in order to avoid immunological reactions inthe patients.

[0013] Need to test each patient for a possible allergy to the animalcollagen.

[0014] Impossibility of treating allergic individuals.

[0015] The globin remains, however, soluble at acidic or basic pHs and,under these conditions, can be sterilely filtered through porousmembranes. For suitable concentrations of 20 to 300 mg/ml, suchsolutions can be treated like protein solutions and make it possible toprepare products such as: sponges, films or granules, using or combiningthe techniques of drying, lyophilization, crosslinking andprecipitation. Some examples are developed below.

[0016] Globin is easy to purify from human red blood cells. The latterare available in sufficient amount from donations which have passedtheir shelf-life, remaining in stock in blood transfusion centers andfor which all the prior health tests were carried out at the time thesample was taken. The preparation of insoluble, injectable globin or ofother globin-based biomaterials therefore represents novel biomedicalapplications making it possible to recover blood donations which havepassed their shelf-life, and to avoid or decrease their destruction.

[0017] The invention can also be implemented- using a blood sample, ofapproximately 5 to 100 ml, taken from the patient to be treated, andconverting it into autologous globin with the same methods as for largevolumes, and then injecting the paste obtained, for the correction ofwrinkles in the same patient or other applications. The number ofsyringes prepared using a sample from the patient may be considerableand may allow the patient to be treated for several years.

[0018] Similarly, human placenta, which is delivered after the birth,contains blood which is generally destroyed by incineration, but whichcan also be used for the invention.

[0019] Bags of blood from donors are officially controlled by the bloodtransfusion organizations, by virtue of biochemical, bacteriological andserological examinations and screening tests for the various viruses andother infectious agents. In the case of placental blood, it wouldobviously be necessary to carry out the same examinations on bloodsamples from the umbilical cord or from the mother, before being able tocollect, conserve and extract the blood from this starting material. Forthe autologous blood, the tests to be carried out can be simplified.

[0020] The implementation of the invention first requires the harvestingand purifying of the red blood cells in these blood samples, or bloodfluids, by simple operations which are already known. The red bloodcells are recovered by low-speed centrifugation. The plasma supernatantis separated and replaced with a physiological saline liquid of PBStype, containing 9 g/l of NaCl and buffered at neutral pH.

[0021] After several washes (3 to 5), the plasma proteins are thusremoved from the red blood cell suspension. One or two volume(s) ofdistilled water is (are) added to the pellet of purified red blood cellsin order to perform an osmotic shock which results in lysis of the bloodcell membranes and releases the hemoglobin in solution, concentrated andpurified. A high-speed centrifugation step (10 to 20 000 rpm) makes itpossible to remove the membrane and cell debris in the pellet. A finalstep consisting in filtration of the supernatant through a membrane witha porosity of 0.2 micron makes it possible to prepare a purified andsterile hemoglobin solution free of particles and derivatives of tissue,cell or membrane origin.

[0022] Heme-globin cleavage at acid pH was described in the presence ofalcohol by Schulz as early as 1898. Anson and Mirsky in 1930, and thenRossi-Fanelli et al., in 1958, used acetone in the presence of acid at0° C. Teale (1957) preferred the use of methyl ethyl ketone in place ofthe acetone. Autio et al. (1984) separated the globin at acid pH byvirtue of absorption and precipitation of the heme with solublecarboxymethyl-cellulose. The globin thus prepared is soluble at acid oralkaline pH, but becomes insoluble as soon as the pH of the aqueoussolution is neutralized to between pH 6 and 8.

[0023] Solubilization experiments at neutral pH were carried out byStrumia et al., in 1951 and 1952, using a prolonged alkaline treatmentwhich results in partial deamidation of the globin at the asparagine andglutamine residues, converted respectively to aspartic acid and glutamicacid (Vars, 1952). Other solubilization experiments were carried out byVolckmann in 1988, by succinylation.

[0024] The insoluble nature in physiological medium explains thepersistence of the globin after tissue implantation, which also makes itresistant to enzymatic degradation, especially if the amount injected isconsiderable, which is the case in filling or tissue augmentationapplications.

[0025] On the other hand, most other proteins can only be precipitatedby high concentrations of salts or of alcohol, which will make theirprecipitates non-biocompatible and non-usable for intra-tissueinjections. In addition, such implants will disappear very rapidly by-diffusion of the precipitating agents and gradual dissolving of theprecipitate on contact with the physiological medium of the tissues.

[0026] The advantage of the invention can be readily verified using apreparation of rabbit globin. The physiological, precipitated globinpaste thus prepared can be injected subcutaneously at various places onthe back or the side of the rabbit. It is easy to verify the innocuityby the absence of local erythema. The persistence of the product underthe skin can be observed by palpation as a function of time. The absenceof antigenic capacity of the product can be verified by a subcutaneousand intramuscular immunization of rabbits, with or without Freund'sadjuvant. Blood samples taken after the immunization make it possible toverify the absence of anti-globin or anti-hemoglobin antibodies usingthe conventional control tests.

EXAMPLES OF PRODUCTION OF PRODUCTS ACCORDING TO THE INVENTION

[0027] Preparation of Rabbit Globin

[0028] Five anesthetized rabbits are bled by cardiac puncture. The bloodis recovered in the presence of heparin or in the presence of sodiumcitrate so as to avoid clotting thereof. 210 ml of blood are thusobtained, which are centrifuged for 30 minutes at 2500 rpm. Thesupernatant containing the plasma is removed with a pipette and thepellet is washed 5 times with 3 volumes of PBS buffer, containing 9 g/lNaCl and buffered at pH 7.2. An equal volume of distilled water isadded, with stirring, to the washed final pellet in order to lyse thered blood cells. The final suspension is centrifuged at 12 000 rpm inorder to remove cell and membrane debris. The supernatant is filteredthrough a cellulose acetate membrane with a porosity of 0.22 micron. 82ml containing 97 g/l of hemoglobin are obtained.

[0029] This solution is poured, with stirring, into 275 ml of 96%ethanol containing 1 ml of concentrated HCl. The final concentration is74% of ethanol and 22 g/l of hemoglobin at acid pH. 3 g of CECA L4Sactive charcoal are added with vigorous stirring for 15 minutes at 4° C.

[0030] The suspension is centrifuged at 15 000 rpm for 30 minutes inorder to remove the charcoal in the form of a pellet. The supernatantcontaining the decolorized acid globin is filtered through a series ofporous membranes, to the smallest porosity (0.2 micron), in order toremove the fine particles of charcoal. The filtrate is diluted with anequal volume of distilled water, the pH is adjusted to 7.4 by additionof NaOH, and the globin precipitates en masse. After 15 hours, theglobin precipitate is recovered by centrifugation, and then washed twicewith 3 volumes of PBS physiological saline containing 9 g/l NaCl andbuffered to pH 7.4. 58.2 g of globin precipitate are harvested at pH7.4. The precipitate is homogenized by successive transferring betweentwo syringes with a volume of 5 ml, linked via a connector with an innerdiameter of 1 to 2 mm, by successively pushing the plunger of eachsyringe so as to cause the precipitate to pass into the other syringe.

[0031] Finally, the homogenized precipitate is distributed into 1 mlsyringes. It is possible to expel the precipitated globin paste from thesyringe, through needles for intradermal injection. The concentration ofglobin in the paste can be adjusted to values of between 30 and 150mg/g.

[0032] Preparation of Human Globin

[0033] 200 ml of human blood which has passed its shelf-life, taken onsodium citrate, are centrifuged for 30 min at 2500 rpm. The supernatantcontaining the plasma is removed with a pipette, also taking up bysuction the superficial whitish cell layer corresponding to theleukocytes. The pellet of red blood cells is washed 5 times with 3volumes of PBS physiological saline containing 9 g/l NaCl and bufferedat pH 7.2, by successive centrifugations. 2 volumes of distilled waterare added to the final pellet in order to lyse the red blood cells. Thehemolyzed suspension is clarified by centrifugation for 30 min at 12 000rpm and filtered through a membrane with a porosity of 0.2 micron. 210ml containing 52 g/l of hemoglobin are obtained, which are conserved at4° C.

[0034] An equal volume of 210 ml of 0.1 N HCl at 4° C. is added, and theentire mixture is poured into 4 l of acetone containing 40 ml of 1 NHCl. The suspension is stirred vigorously and left to stand for one hourat ambient temperature, under a chemical hood. The heme dissolved in theacetone is removed by filtration through porous cloth and the globinprecipitate is recovered, washed in acidic acetone and dried under astream of air. The globin is redissolved in aqueous solution at pH 2 andcan be precipitated again with acid acetone to complete decoloration ofthe globin.

[0035] Finally, the aqueous acid globin solution is filtered sterilelythrough a membrane with a porosity of 0.2 micron, and then precipitatedby neutralization of the pH by adding NaOH until a pH of 7.4 isobtained. Syringes of globin paste precipitated at neutral pH can beprepared as in the preceding example. The operation to neutralize theacidic globin solution can be carried out by adding sodium hyaluronateat alkaline pH. In this case, there is formation of a paste of insolubleglobin complexed and impregnated with the hyaluronate, providing alubricating function which improves the injectable nature through fineneedles.

[0036] Other Preparation of Human Globin

[0037] The process of Example 1 is carried out using a controlled bloodcell pellet which has passed its shelf-life, obtained from a bloodtransfusion center. Syringes containing a paste of precipitated humanglobin which is biocompatible and implantable by injection are obtained.

[0038] Preparation of Human Globin Having Undergone Alkaline Treatmentwith 0.1 or 1 M Sodium Hydroxide for 1 Hour at 20° C.

[0039] The process of Example 1 or 2 is carried out until the globinprecipitate is obtained at pH 7.4, before washes. This precipitate isdissolved, again, in 3 volumes of 0.1 M to 1 M NaOH at 20° C. for onehour, with stirring.

[0040] The solution is then neutralized by the addition of an equalvolume of HCl of the same molarity and the pH of the suspension isadjusted between and 7 and 7.4. The globin precipitate is then harvestedby centrifugation and then washed in PBS physiological saline as in thepreceding examples. The precipitated globin paste, to which hyaluronicacid, may have been added, is distributed into syringes and theinjectable nature of the product obtained is again verified. Thisalkaline treatment of the globin makes it possible to improve the healthsafety guarantees for the product without significantly modifying theinsoluble nature of the globin at neutral pH.

[0041] Preparation of a Paste of Precipitated andGlutaraldehyde-Crosslinked Globin

[0042] This treatment is possible in order to increase the implantresorption time. The final globin precipitate is suspended at 2% in PBS.Glutaraldehyde is added, with stirring, at a concentration of 1 mg/g ofprecipitate. After incubation for 1 hour at 20° C., the globinprecipitate is washed and placed in syringes as in the precedingexamples.

[0043] Other crosslinking agents such as dialdehydes or polyaldehydescan be used, in particular polysaccharides oxidized with periodic acid,such as oxidized dextran, oxidized starch, or oxidized hyaluronic acid.

[0044] Preparation of Syringes of Sterile Precipitated Globin Paste

[0045] To prepare sterile syringes, it is necessary to work underaseptic conditions as soon as sterilizing filtration of the acidicglobin solution through a membrane with a porosity of 0.2 micron hastaken place. This can be done under a laminar flow hood in a class 100or 1000 sterile zone, or with a sterile chamber, accessible from theoutside via flexible latex gloves. The operations of precipitation andcentrifugation of the precipitate should be performed in centrifugationbuckets which have been sterilized and wrapped in a protective film.

[0046] Another method consists in distributing an acidic solution ofsoluble globin, which has been sterilely filtered, into a first syringeand a second alkaline solution, which has been sterilely filtered, intoa second syringe. The pH of each syringe is adjusted in such a way thatthe subsequent mixture thereof is at neutral pH. The linking of thesetwo syringes, by virtue of a sterile connector, makes it possible toproduce a sterile homogeneous mixture of neutral pH, via successivetransfers from one syringe to the other. A paste is obtained byspontaneous precipitation of the globin. The addition of sodiumhyaluronate to the alkaline syringe makes it possible to incorporatehyaluronate into the final globin. When the second alkaline syringe doesnot contain globin, it is possible to incorporate a crosslinking agenttherein.

[0047] Final Sterilization of the Syringes of Precipitated Globin Paste

[0048] A sterilization of the syringes can be carried out by irradiationat a dose of between 5 and 30 kilogray.

[0049] Production of an Insoluble Gel or Film from Unmodified SolubleGlobin

[0050] A solution of soluble globin is prepared by dissolving theacetone-based globin powder at pH 3, at a concentration of 20 to 120mg/ml, in aqueous solution. This solution is sterilely filtered througha membrane with a porosity of 0.2 p, and then adjusted to pH 5 by addingsterile 1 N NaOH with stirring.

[0051] Oxidized starch at pH 3.5, or another aldehyde, polyaldehyde orother crosslinking agent is added to the mixture at a concentration of0.5% with stirring for 5 min. The sterile mixture is poured over a flatsurface in order to obtain a thickness of 1 to 3 mm of liquid, at atemperature of 20 to 37° C., under a laminar flow.

[0052] The liquid product gradually gels by virtue of the crosslinkingof the globin chains, induced by the oxidized starch, and thendehydrates under the stream of air, if it is desired to obtain a film.

[0053] The final film, at a thickness of between 20 and 200 μ, accordingto the initial concentration of material, can be sterilized by beta- orgamma-irradiation or with ethylene oxide. Well-known film-forming agentscan be added, such as hyaluronic acid, other polysaccharides ormucopolysaccharides, polyethylene glycol, glycerol, etc. Such anadditive makes it possible to give the film flexibility and strength.Such a film can be used alone to protect a wound or can be associatedwith various prostheses (vascular prostheses, strengthening lattices,porous matrices) in order to make them impermeable, improve theirbiocompatibility, or confer anti-adhesion properties or an adhesivenature, according to known techniques already used with other products.

[0054] In a variant, it is possible to produce a film from globinsoluble at pH 5 as previously, but without introducing any crosslinkingagent. The final crosslinking of the dried film is performed by thefinal irradiation which creates covalent bonds between the globinchains.

[0055] Bibliography

[0056] ANSON M. L.—MIRSKY A. E. (1930)

[0057] Protein Coagulation and its reversal. The preparation ofinsoluble globin, soluble globin and heme.

[0058] J. Gen. Physiol. 13, 469-476

[0059] AUTIO K—KIESVAARA M.—MALKKI Y.—KANKU S. (1984)

[0060] Chemical and functional properties of blood globin prepared by anew method

[0061] Journal of Food Science 49, 859-862

[0062] ROSSI-FANELLI A.—ANTONINI E.—CAPUTO A. (1958)

[0063] Studies on the structure of haemoglobin

[0064] I-Physicochemical properties of human globin

[0065] Biochem. Biophys. Acta 30, 608-615

[0066] SCHULZ F. N. (1898)

[0067] Der Eiweisskörper des hämoglobins [The protein body ofhemoglobin]

[0068] Ztsch. F. physiol. Chem. 24, 449-460

[0069] STRUMIA M. M.—SAMPLE A. B.—MAWR B. (1951)

[0070] Modified globin

[0071] I-Method for preparation from human erythrocytes.

[0072] J. Lab. and Clin. Med. 37, 959-968

[0073] STRUMIA M. M.—McGRAW J. J.—SAMPLE A. B.—MAWR B. (1952)

[0074] Modifed globin

[0075] IV-Some of the physiological properties of modified human globin

[0076] J. Lab. and Clin. Med. 40, 2, 211-222

[0077] TEALE F. W. J. (1957)

[0078] Cleavage of the haem-protein link by acid methyl-ethyl keton

[0079] Biochem. Biophys. Acta 26, 437

[0080] VARS H. M.—BOXER G. E.—MAWR B. (1952)

[0081] Modified Globin

[0082] II-Chemical changes in human globin by alkaline modification

[0083] J. Lab. and Clin. Med. 39, 5, 743-751

[0084] VOLCKMANN H. (1988)

[0085] Essais de développement d'un substitut plasmatique d'origineplacentaire [Attempts to develop a plasma substitute of placentalorigin]

[0086] These d'ingénieur [Engineer's thesis] CNAM-Lyon

1. A preparation that can be injected or implanted into the human oranimal body, which comprises, as main component, globin that isinsoluble at physiological pH, biocompatible and sterile.
 2. Thepreparation as claimed in claim 1, wherein the globin is a globin ofhuman origin.
 3. The preparation as claimed in claim 1, wherein theglobin in the preparation is in the precipitated state.
 4. Thebiocompatible injectable preparation as claimed in claim 1, whichcomprises homogenized globin.
 5. The preparation as claimed in claim 4,wherein the globin is in the form of an injectable homogenized paste. 6.The preparation as claimed in claim 4, wherein the homogenized paste canbe injected through a hypodermic needle.
 7. The preparation as claimedin claim 4, wherein the concentration of globin in the injectablepreparation is between 30 and 150 mg/g.
 8. The preparation as claimed inclaim 4, wherein the pH of the preparation is between 6 and
 8. 9. Thepreparation as claimed in claim 1, wherein the globin is in suspension.10. The preparation as claimed in claim 1, wherein the globin is insolution, at a pH below 6 or above 8, in a pharmaceutically acceptableliquid vehicle.
 11. The preparation as claimed in claims 1, wherein theglobin is present in the preparation in the form of a gel.
 12. Thepreparation as claimed in claim 1, which also comprises a lubricant. 13.The preparation as claimed in claim 12, wherein this lubricant is chosenfrom solutions of triglycerides, of polyethylene glycol, of hyaluronate,of hyaluronic acid, of oxidized cellulose, or of polysaccharides or ofmucopolysaccharides.
 14. The preparation as claimed in claim 1, whichcomprises a crosslinking agent.
 15. The preparation as claimed in claim14, wherein the crosslinking agent is chosen from glutaraldehyde,dialdehydes and polyaldehydes, in particular polysaccharides oxidizedwith periodic acid, including oxidized dextran, oxidized starch oroxidized hyaluronic acid.
 16. The preparation as claimed in claim 1,which comprises or consists of a globin film, it being possible for thepreparation to optionally contain a film-forming agent, in particularsuch as collagen, gelatin, hyaluronic acid, oxidized cellulose,polyethylene glycol or glycerol.
 17. The preparation as claimed in claim16, wherein the film has been obtained by dehydration of a gel or of asolution.
 18. The preparation as claimed in claim 1, which is producedin the form of a solid implant.
 19. The preparation as claimed in claim16, which is crosslinked.
 20. The preparation as claimed in claim 19,which is crosslinked by the addition of a crosslinking agent and/or byirradiation.
 21. The preparation as claimed in claim 1, which alsocontains at least one of the following active principles: healingproduct, growth factor, antibiotic.
 22. The preparation as claimed inclaim 1, which contains cells, in particular cells cultured using theglobin of the preparation as culture support, before injection orimplantation, which cells can in particular be skin fibroblasts,chondrocytes, osteoblasts or stem cells.
 23. The use of a preparation asclaimed in claim 1, for producing a tissue filling material.
 24. The useas claimed in claim 23, for filling skin cavities, wrinkles or scars,and bone or cartilage cavities and fractures.
 25. The use of apreparation as claimed in claim 1, for increasing tissue volume.
 26. Theuse as claimed in claim 25, which is provided for augmenting sphincters,in particular urinary or digestive sphincters, or vocal cords.
 27. Theuse of a preparation as claimed in claim 16, for producing films and/orcompresses for the protection and/or separation of surgical ornonsurgical, external or internal wounds or scars.